Ion AmpliSeq™ panel design and CNV detection
Copy Number Variants (CNVs) can be detected using Ready-to-Use and Custom Ion AmpliSeq™ panels and the Ion Reporter™ Software. The following panels support CNV detection in Ion Reporter™ Software:
Use following guidelines to design a custom Ion AmpliSeq™ panel for robust CNV detection in Ion Reporter™ Software:
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We have verified that CNVs can be called with Ion AmpliSeq™ panels as small as 200 amplicons. Smaller panels have not yet been tested.
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The algorithm used by Ion Reporter™ Software should be robust if no more than 20% of amplicons in a panel have a CNV. So, if you are interested in whether a gene is affected by a CNV, there should typically be at least 5 genes in the panel, only one of which has a CNV in any sample. If more than 20% of the amplicons have a CNV in the panel (for example, in the case of a massively aneuploid tumor sample), the change in copy number can be detected, but the exact copy number value predicted may not be correct. This is because the normal diploid (‘ploidy=2’) state can be hard to identify in such samples, and so CNVs can be called with the wrong copy number.
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Generally, CNVs can be robustly detected if at least 10, and ideally 20, amplicons cover a CNV. It can be possible to call CNVs with fewer amplicons (especially if using the algorithm’s low stringency setting), but if the data is somewhat noisy or if the coverage is low, a CNV covered by fewer than 10 amplicons can be missed.
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When adding amplicons to an existing panel to make it robust for CNV detection (for example, to detect CNVs on a single-gene panel), the extra amplicons should be outside the region with expected (interrogated) CNV. For example, the extra amplicons should be outside the gene and ideally on a different chromosome.
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Putting amplicons on multiple chromosomes can improve robustness. Consider putting at least 2 or more amplicons on any one chromosome (20 on a chromosome to detect aneuploidy on that chromosome).
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Avoid population-based and common non-pathogenic CNV regions when designing amplicon coverage. These regions can be identified using the Database of Genomic Variants or similar resources. When adding amplicons to a panel that contains a single gene or a small numbers of genes, it is best to place extra amplicons farther away from the genes of interest or on different chromosomes, and not immediately flanking the gene of interest. Placing more amplicons immediately flanking the pre-existing amplicons risks not providing the extra “normal” 80% data necessary for CNV detection if a CNV occurring happens to be larger than the gene’s combined coding and flanking areas.
Best practices for CNV detection from Ion AmpliSeq™ sequencing data
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Consistent sample preparation and pooling
It is important for the control and experimental Ion AmpliSeq™ libraries to be prepared using the same procedure and that each pool of a particular library is quantified using the Ion Library TaqMan™ Quantitation Kit (Cat. No. 4468802) before pooling.
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CNVs are reported based on their copy number relative to the control sample used. Therefore, when performing a paired or tumor/normal comparison, it is best practice to select a control sample with no known CNVs in any region covered by the Ion AmpliSeq™ panel used. If most or all test samples are reported as having a CNV in the same region, one possible cause is that the control sample has a CNV in that region.
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CNVs with higher confidence scores are more likely to be true positives. You can filter reported CNVs by confidence scores assigned by the software. By default, Ion Reporter™ Software filters for CNVs with scores >10, indicating high confidence CNVs. However, the exact threshold can depend on the panel and the nature of the CNVs under investigation.
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Copy number state and confidence scores on X and Y chromosomes are determined relative to the gender of the control sample. For example, the expected ploidy of X for a male control sample is 1: if the test sample also has 1 copy of X, the confidence score for a CNV is 0, because there is no evidence of an unexpected ploidy state.
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If the number of copies of a CNV is >10, this is reported as copy number of 10 in Ion Reporter™ Software.